The present invention generally relates to localization methods, systems, and computer program products, such as for use in imaging applications, and in particular to methods, systems, and computer program products for localizing photons and light sources emitting photons with high spatial and temporal resolution.
The ability to image and track individual macromolecules labeled with either single fluorophore molecules or sub-resolution fluorescent nano-particles with high spatial precision has permitted testing hypotheses concerning intra-molecular conformational changes that underlie biological processes both in vitro and in vivo. Such measurements may permit the testing of hypotheses concerning the specific intra-molecular conformational changes that underlie important biological processes.
The majority of studies involving dynamic tracking of sub-resolution particles have used conventional wide-field microscope systems and, most often, wide-field microscope systems combined with total internal reflectance fluorescence (TIRF). However, there are circumstances where confocal (single- and multi-photon) scanning microscope systems would provide additional technical advantages, such as background noise rejection permitting imaging deeper into specimens. In particular, laser scanning confocal microscopes are capable of imaging single fluorophore molecules and sub-resolution fluorescent nano-particles as diffraction limited single point sources of light. However, the efficacy of these studies depends on the precision with which the location of a sub-resolution fluorescent label imaged as a diffraction-limited single source of light can be measured. Theoretically, this location can be determined with arbitrary precision as the center of a diffraction-limited spot, but practical localization precision depends on the number of photons available to form an image.
Standard statistical curve-fitting methods for establishing the (x,y) location of a point source of light in a plane, such as to localize a sub-resolution particle, involve fitting a Gaussian intensity profile to an image of the point source. The centroid or maximum of the fitted Gaussian represents the (x,y) location of the particle. The confidence with which the (x,y) position is known depends on, among other factors, the width of the optical point spread function (PSF) of the microscope system and the number of photons collected to form the image. Confidence in the knowledge of the (x,y) position is adversely affected by several sources of uncertainty, namely those associated with photon noise, background noise, and pixel size.
Standard statistical methods work sufficiently well for wide-field microscope systems. However, due primarily to the binary nature of the photon position map, standard statistical methods are inadequate to localize the spatial coordinates for the origin of a photon acquired by a scanning microscope. Thus, there is a need for improved methods to localize the position of a photon in the binary photon position map.